Protein A-based antibody immobilization onto polymeric microdevices for enhanced sensitivity of enzyme-linked immunosorbent assay.
Highly efficient antibody immobilization is extremely crucial for the development of high-performance polymeric microdevices for enzyme-linked immunosorbent assay (ELISA). In this article, a site-selective tyrosinase (TR)-catalyzed protein A strategy for antibody immobilization was developed to enhance the sensitivity of ELISA in poly-(methyl methacrylate) (PMMA) microchannels for interferon-gamma (IFN-gamma) assay. To effectively immobilize the target antibodies, oxygen plasma was first used to activate the inert PMMA. This is followed by poly(ethyleneimine) (PEI) coating, an amine-containing functional polymer. For comparison, protein A was also immobilized through the commonly used amine-glutaraldehyde (GA) chemistry. Oxygen plasma treatment effectively increased the amount of PEI attachment and subsequent binding efficiency of the primary antibody. The antibody immobilized via TR-catalyzed protein A was able to provide much better specific antigen capture efficiency than GA chemistry due to the optimal spacing and orientation. Consequently, by using this new method, the detection signal and the signal-to-noise ratio of the ELISA immunoassay in microdevices were all significantly improved. In comparison to the standard assay carried out in the 96-well microtiter plate, the treated microchannels exhibited a broader detection range and a shorter detection time. And the detection limit was also decreased to 20 pg/mL, much lower than that obtained in other microdevices.
Yuan
Yuan
Y
Department of Chemical and Biomolecular Engineering, NSF Nanoscale Science and Engineering Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210, USA.
He
Hongyan
H
Lee
L James
LJ
D055910:Antibodies, Immobilized; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007097:Imines / Q000737:chemistry; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D008832:Microchemistry / Q000379:methods*; D046208:Microchip Analytical Procedures / Q000379:methods; D018625:Microscopy, Atomic Force; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011095:Polyethylenes / Q000737:chemistry; D019904:Polymethyl Methacrylate / Q000737:chemistry; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A
D055910:Antibodies, Immobilized; D007097:Imines; D011095:Polyethylenes; D013205:Staphylococcal Protein A; C505405:poly(ethylene imine); D007371:Interferon-gamma; D019904:Polymethyl Methacrylate; D014442:Monophenol Monooxygenase; D010100:Oxygen